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. Author manuscript; available in PMC: 2023 Oct 1.
Published in final edited form as: Clin Pharmacol Ther. 2022 May 31;112(4):808–816. doi: 10.1002/cpt.2637

Figure 1. Proposed scheme of molybdenum cofactor (Moco) catabolism and urothione-synthesizing activity in mouse crude protein extracts.

Figure 1.

(A) The pathway involves at least two enzyme-catalyzed reactions methylation and dephosphorylation. The order of both reactions is unknown. Also, release of molybdenum, pyran ring opening and pterin oxidation, are also required for the formation of urothione and believed to take place in early steps of this pathway. Moco [1], a methylated intermediate [2], urothione [3], and Form B [4], the air oxidized product of Moco are shown. (B) HPLC analysis of in vitro generated urothione from mouse liver extract in the presence (black) and absence (gray) of SAM. (C) In vitro urothione synthesis using crude protein extracts (4 μg/μL) from liver, kidney and brain using in vitro synthesized thiopterin (2.72 μM) in the presence of 50 μM SAM. (D and E) Purification of urothione synthesizing activity from mice crude liver extracts using anion exchange and size exclusion chromatography. Fractions were taken and urothione synthesis was determined using in vitro synthesized molybdopterin (MPT) in the absence (D) and presence of alkaline phosphatase (E). Gray highlight indicates the fraction which was further purified (D) or analyzed for protein content (E). (F) Results of peptide mass finger printing derived from the sample with highest activity shown in panel E. (G) HPLC analysis of in vitro generated urothione from mouse liver extract in the presence of SAM (black) or betaine (gray).